Biochem. J. (1992) 282, 601-604 (Printed in Great Britain)
601
A study of Ca2+-heparin complex-formation by polarimetry
David GRANT, William F. LONG,* Colin F. MOFFAT and Frank B. WILLIAMSON
Polysaccharide Research Group, Department of Molecular and Cell Biology, University of Aberdeen, Marischal College,
Aberdeen AB9 lAS, Scotland, U.K.
Possible inflexions in isothermal binding plots derived from equilibrium-dialysis measurements and in equivalent plots
obtained by polarimetric measurements accord with the possibility that discrete Ca2+-heparin-water complexes/phases
may exist, the nature and proportions of which depend on the conditions under which the interaction occurs. Analysis
of the plots obtained by polarimetric study of chemically modified heparins suggests, for individual substituents groups,
an order of importance of carboxylate > N-sulphonate > N-acetyl > 0-sulphate for the Ca2+-heparin interaction
occurring at [Ca2+]/[heparin disaccharide] ratios of less than 0.5. At higher ratios, transitions occur that eventually lead
to the formation of a complex in which the stoichiometry of association is 1 Ca2` ion/heparin disaccharide unit.
INTRODUCTION
The present paper explores Ca2+-heparin interaction with the
of equilibrium dialysis, polarimetry and i.r. spectroscopy; the
results obtained accord with the notion that more than one type
of Ca2+-heparin complex may exist.
use
Heparins are strongly acidic and possess several chemical
that interact with a variety of cations. Many modulations
by metal ions, including Ca2l, of the activities of heparins in vivo
and in vitro have been discussed (e.g. Long & Williamson, 1979,
1982). Results of heparin-cation studies carried out with dilute
aqueous solutions have been interpreted either in terms of simple
electrostatic interactions or in terms of additional chemical
interactions resulting in cations binding to specific sites on the
polymer. Manning (1969a,b,c) predicted that counter-cation
condensation on to a polyanion occurred whenever a critical
density of charge along the polymer was exceeded, and suggested
the existence of a critical counterion concentration, below which
condensation occurred, and above which the ions existed in an
ion atmosphere. An additional, 'two-variable' model (Manning,
1977, 1978) extended the idea to include the possibility of
exchange of cations between sites. In these models, ion interaction
is independent of the chemical nature of the anionic sites on the
polyelectrolyte, and, except for its charge, is independent of the
chemical nature of the counterion.
Physical studies by Boyd et al. (1980), Liang & Chakrabarti
(1982) and Mattai & Kwak (1988), however, support the concept
of site-specific Ca2+-heparin interaction. I.r. spectra of different
cation-heparin complexes showed cation-dependent variation
and a cation-dependent transition between conformational
states, possibly with different hydration chemistries (Grant et al.,
1987a,b). Further, a dependence on [Na+] of the diffusion rates of
ions in aqueous heparin solutions lacking additional electrolyte
(Ander & Lubas, 1981 ; Ander & Kardan, 1984), and an apparent
polyanionic charge on heparin smaller than that predicted by
electrostatic theory (Tivant et al., 1983), suggest that all heparincation interactions may not be explained by simple electrostatics.
These studies are well summarized by Nieduszynski (1989).
For Ca2+ binding, a predominant role for carboxylate groups
has been suggested (Grant et al., 1987a), with N-sulphonate
groups playing a subsidiary role (Liang & Chakrabarti, 1982;
Ayotte & Perlin, 1986). In addition, in the presence of Ca2+ the
polymer may be held in a conformation that is stabilized by
an interaction between N-sulphonate and carboxylate groups,
possibly involving hydrogen-bonding (Grant et al., 1987a,b).
Additional complications involve the possibility of multiphasic
interactions (Burger et al., 1984; Grant et al., 1986), and the
involvement of co-ordinated water molecules in the interaction
(Grant et al., 1987a).
groups
*
To whom correspondence should be addressed.
Vol. 282
EXPERIMENTAL
The source, preparation and properties of the heparin and of
the chemically modified heparins used have been described
previously (Grant et al., 1987a, 1989). Polymers were converted
into particular cation complexes by passage through appropriate
cation forms of Amberlite IR-120 cation-exchange resin. Sparksource m.s. showed a greater than 99 % efficiency of the cationexchange process. Water contents of Na+ forms of heparin and
of modified heparins were deduced following calculation of
formula weights from uronic acid residue (Blumenkrantz &
Asboe-Hansen, 1973) or, for carboxy-reduced heparin, glucosamine residue (Hurst & Settine, 1981) assay. An average heparin
disaccharide unit was taken to be hexadecahydrated (Atkins
et al., 1974; Grant et al., 1990) tetrasodium 2-0-sulphatoiduronosyl 6-0-sulphatoglucosamine 2-N-sulphamate.
Details of equilibrium dialysis were as described by Grant
et al. (1986) and Woodhead et al. (1986). I.r. spectroscopy of
aqueous films of heparin-cation complexes was carried out by a
multiple-specular-reflectance technique as described by Grant
et al. (1987a).
Polarimetric measurements were made with a Thorn
Automation-NPL type 243 automatic polarimeter. Nitrocellulose
membrane (2 ,m)-filtered solutions of heparin or of modified
heparins (6 ml in water, except for the experiment reported in
Fig. 1, in which the solvent was 0.15 M-NaCl) were prepared and
placed in the silanized glass cell immediately before the experiment. Consecutive 1 ,l additions of CaCl2 solution (2.02 M)
were made to the cell. Correlations were applied for the cell
blank and for the small dilution effects resulting from CaCI2
addition. Addition of CaCl2 solution, which itself was not
optically active, elicited an increase in optical rotation that
reached a maximum at high [Ca2+] (Moffat et al., 1984). Similar
experiments were carried out with solutions of LiCl, NaCl,
CuCl2 and ZnCI2. Additions of each of these to the polymer
solution elicited an increase in polymer optical rotation; in the
case of CuCI2 solution, addition produced a decrease in optical
rotation. Addition of CaC12 solution to modified heparins
produced an increase in polymer optical rotation, except for deN-sulphonated de-0-sulphated N-acetylated heparin, in which
D. Grant and others
602
case a decrease in optical rotation occurred. For the purpose of
comparing results from polarimetric measurements with those
obtained from equilibrium dialysis experiments, the change in
optical rotation that occurred after addition of cation was
expressed as [(optical rotation in the presence of cation) - (optical
rotation in the absence of cation)]/[(maximal optical rotation at
high cation concentration) - (optical rotation in the absence of
cation)]. At 'high' cation concentration, the [cation]/[heparin
disaccharide] ratio was 6; at this ratio, no further detectable
increase in the corrected optical rotation value occurred on
addition of more cation. Optical rotations were recorded with
light of 546 nm wavelength in a pathlength of 0.61 dm, at a
temperature of 25 °C, and at a polymer concentration that, if not
indicated otherwise in Fig. 3, was 0.477 g/100 ml in water.
Because the different degrees of hydration of heparin and
modified heparins (Table 1) contribute to polymer mass, but are
unlikely to contribute directly to polymer optical activity, experimentally recorded optical rotations are reported directly,
rather than as specific rotations.
RESULTS AND DISCUSSION
Fig. 1 (O symbols) shows the isothermal saturation fractions
of potential binding sites on heparin occupied by Ca2" as a
function of [total Ca2"], when a binding site is taken to be an
average heparin disaccharide unit. The results are from an
equilibrium-dialysis experiment. The * symbols in Fig. 1 show
an equivalent plot derived from optical-rotation changes
observed under equivalent conditions of [heparin] and [NaClI].
Fig. 2 shows a plot of the maximal increase or decrease in optical
rotation (obtained when high [cation] was added to solutions of
Na+-heparin), against independently derived association constants for heparin-cation interactions. The relationship apparent
in Fig. 2, together with the near-identity of the plots in Fig. 1,
suggest that optical-rotation measurements offer an indirect but
1.2
*
0
e
0
0
1.0
0
0
C
O
0
0
0
.12C)
0.8
0e
0
+
0.6
0 0
0
E
a)
0
0.4
0
0
0
U,
0.2
0
*0
0
10
20
30
[Ca2+1 (mM)
Fig. 1. Isothermal saturation fractions of binding sites on heparin occupied
by Ca2", and equivalent fractions derived from polarimetric measurements, as functions of Itotal Ca2+j
A binding site was taken to be an average heparin disaccharide unit
(see the Ex$perimental section). - Isothermal saturation fractions
obtained by equilibrium dialysis; 0, equivalent fractions derived
from polarimetric measurements as described in the Experimental
section. In both experiments [heparin disaccharide unit] was 0.005 M
and [NaClI] was 0. 15 M.
C
o
°°-
Cu2+
0.04
0
0.030
m 0.02
Ca 2+
C
Zn
0.01
.E_
*Li+
x
*Na+
500
1500
2000
1000
Association constant (M-1)
Fig. 2. Maximal alterations in optical rotation seen on addition of cations
to Na'-heparin as functions of independently derived cation-heparin
association constants
Heparin association constants were from the following sources:
Herwats et al. (1977), Li', Na', Cu2+; Mattai & Kwak (1981), Ca21;
Woodhead et al. (1986), Zn2+. In each case optical rotations were
measured at a [cation]/[heparin disaccharide unit] ratio of 6.
0
simple, fast and non-destructive method of exploring heparincation association. For example, from the plot in Fig. 2, tentative
association constants for the interaction of Mg2+, K+, Fe 2+ and
Ba42+ with heparin under these conditions may be estimated to
be approx. 25, 75, 555 and 600 M-1 respectively. These values
accord with previous estimates, where they are available, of the
affinities of these cations for heparin (Dunstone, 1962).
Fig. 3 shows polarimetry-derived plots for Ca 2+-heparin
interaction obtained with various concentrations of heparin in
water rather than, as in Fig. 1, salt solution. They suggest that,
under these conditions, optical-rotation changes approach completion at a [Ca 2+]/[heparin disaccharide] ratio of about 1.
Inflexions in similar plots derived from equilibrium-dialysis
experiments have been interpreted in terms of a multiphasic
interaction between cation and polymer (Boyd et al., 1980;
Grant et al., 1986; Woodhead et al., 1986). Future polarimetric
measurement, with even more precisely structurally defined
polymer preparations than are used here, offers the possibility of
more detailed analysis of putative points of inflexion than has
been undertaken following equilibrium-dialysis experiments, in
which the limited number of samples taken can restrict the
precision of the plots constructed. However, the present results
suggest that an inflexion may occur at a ratio of about 0.5;
certainly at lesser ratios a direct linear relationship appears to
exist between the polarimetry-derived fraction and the [Ca2+]/
[heparin disaccharide] ratio. This indicates that, under such
conditions, cation-heparin interaction occurs by a mechanism
that cannot be described adequately in terms of conventional
solution-phase reversible-equilibrium thermodynamics. This is
also suggested by the coincidence of the plots obtained with
different heparin concentrations (Fig. 3).
Because of the relationship suggested by the results in Fig. 2,
polarimetry-derived plots for the interaction of Ca 2+ with several
modified heparins were prepared, in an attempt to assess the
importance of various heparin substituent groups to Ca22+ binding. Table 1 shows maximal optical-rotation changes seen when
high [Ca22+] was added to solutions of Na+ forms of the polymers.
The results suggest that each of the modifications decreases the
water component of the Na'-polymer complex. Fig. 4, in which
the change in optical rotation on addition of high [Ca22+] is
plotted against the water component of the polymers, indicates
that the magnitude of the change may depend on the number of
1992
Ca2+-heparin interaction
603
I
I*
c
1.0k
*IS3
°0
0
coo
18
0)az
:2 16
6D
5 14
0
.u) 12
a)
0.8
4)
E
75 8
L: 6
0)
oc
0)
QO
0
C
c
._
*
0.6~
(0
o
'
0
0
c
0.4
S
S
C
0
0.020
0.010
Maximal change in optical rotation (°)
Fig. 4. Maximal optical-rotation changes occurring on addition of Ca"+ to
Na+ forms of modified heparins as a function of polymer hydration
Data from which the graph is constructed are given in Table 1.
8
0
a,Q
0
C)
(3,
Cu
ED
0
ED 10
F
F
-8
a
0.2 j
0
.0
t
0
0.5
1.0
1.5
3.0
2.5
2.0
lCa2+l/Mheparin disaccharide unit]
Fig. 3. Polarimetry-derived fraction of binding sites on he
e
Ca"+ as a function of ICa'+I/Iheparin disaccharidde unit
A binding site was taken to be an average heparin di:saccharide unit
(see the Experimental section). Fractions were derive
metric measurements as described in the Experinnental section.
2.38; O,
Na+-heparin concentrations (mg/ml) in water were
4.77; C, 9.56; 0, 14.30.
:t,
water molecules available for release from the pol ymer on cation
binding. Table 1 also shows that, in each case modification
decreases the extent of the optical-rotation charage seen in the
presence of high [Call], suggesting a lowering off the affinity of
the polymer for Ca2". The [Ca2l]/[polymer disaccEharide] ratios at
which a linear to non-linear transition occurred in the plots for
unmodified, de-N-sulphonated, de-N-sulphonated N-acetylated,
de-N-sulphonated de-O-sulphated, de-N-sulphonated de-Osulphated N-acetylated, de-N-sulphonated de-O-sulphated re-Nsulphonated and carboxy-reduced heparins were 0.46 (at a
fraction of 0.54), 0.28 (at a fraction of 0.58), 0.33 (at a fraction
of 0.62), 0.15 (at a fraction of 0.22), 0.33 (at a fraction of 0.59),
0.40 (at a fraction of 0.74) and 0.22 (at a fraction of 0.25)
respectively. This suggests that the particular polymer-Ca2+
complex/phase, formation of which is implied by the existence of
the linear portion of the polarimetry-derived plots, is stable only
at [Ca2+]/[polymer disaccharide] ratios that are lower for the
modified heparins than for the unmodified polymer, and that, at
higher ratios, destabilization of the complex/phase, to generate
the non-linear portion of the plot, occurs. By using the above
values, it is possible to derive the approximate empirical relationship: [Ca2+]/[polymer disaccharide] at the transition
point = (0.20 x carboxylate groups/disaccharide) + (0.18 x Nsulphonate groups/disaccharide) + (0.08 x N-acetyl groups/
disaccharide) + (0.06 x 0-sulphate groups/disaccharide). This
suggests the greater importance of the carboxylate and Nsulphonate groups and lesser importance of the N-acetyl and Osulphate groups in the heparin-Ca2+ interaction indicated by the
linear portion of the polarimetry-derived plots.
Table 1. Maximal optical-rotation changes occurring on addition of Ca2" to Na+ forms of modified heparins
Optical rotations were measured at 546 nm as described in the Experimental section. 'High' [Ca2"] refers to a [Ca2"]/[polymer disaccharide]
ratio of 6.
Optical rotation (0)
Polymer
Unmodified
Carboxy-reduced
De-N-sulphonated,
N-acetylated
De-N-sulphonated,
de-0-sulphated,
re-N-sulphonated
De-N-sulphonated
De-N-sulphonated,
de-0-sulphated
De-N-sulphonated,
de-O-sulphated,
N-acetylated
Vol. 282
Water
molecules/
disaccharide
In the absence
of Ca2"
16
14
13
In the presence
of high
[Ca2"]
Change
0.1548
0.1682
0.1683
0.1769
0.1848
0.1805
0.0221
0.0166
0.0122
12
0.2441
0.2551
0.0110
8
7
0.2048
0.2490
0.2115
0.2542
0.0067
0.0052
7
0.2760
0.2730
0.0030
D. Grant and others
604
*
1450
Mg2+
0
Cu2+
E 1440-
Q
LL
0
1430-
* Ca2+
* Ba2+
X~1 4201410
-r
0
0.03
0.04
0.05
0.01
0.02
Maximal changes in optical rotation (°)
Fig. 5. Frequency of the cation-heparin carboxylate symmetric stretchingbond absorbance as a function of the maximal alteration in heparin
optical rotation on cation addition
Optical rotations were measured at a [cation]/[heparin disaccharide
unit] ratio of 6.
simple aqueous solution equilibrium processes and that are
maintained by forces additional to simple electrostatic ones.
Possible inflexions in isothermal binding plots obtained by
equilibrium dialysis and in equivalent plots obtained by polarimetric measurements accord with the suggestion (Boyd et al.,
1980; Burger et al., 1984; Grant et al., 1986) that multiple
discrete Ca2+-heparin complexes/phases may form, the nature
and proportions of which depend on the conditions in which the
interaction occurs. The present results suggest that when Ca 2+ is
added to Na+-heparin solution Ca2+-heparin complexes are
formed with an initial stoichiometry of 1 Ca2+ ion/heparin
tetrasaccharide unit. Once all of the heparin has been converted
into this form, then further Ca2+ addition results in Ca2+ binding
to form a complex with a stoichiometry of 1 Ca2+ ion/
disaccharide unit. It is possible that further experimentation may
reveal the existence of discrete Ca2+-heparin complexes having
stoichiometries intermediate between 0.5 and I Ca2+ ion/
disaccharide unit.
We thank Dr. N. E. Woodhead for carrying out the equilibrium
dialysis. C. F. M. was supported by a University of Aberdeen Medical
Endowments Studentship.
REFERENCES
Conversion of Na+-heparin into Ca2+-heparin results in a shift
in the frequency at which the heparin carboxylate symmetric
stretching bond absorbs (Grant et al., 1987a), and an increase in
N-sulphonate absorbance at 1185 cm-' (Grant et al., 1987b).
This suggests involvement of these anionic groups in cation
binding. There is little effect on 0-sulphate group absorbance
(Grant et al., 1987a). The conversion of Na+7heparin into
Ca2+-heparin is accompanied by a decrease in polymer water
content (Grant et al., 1990). A near-i.r. spectroscopic analysis of
Ca2+-heparin suggests that, in this form of the polymer, water is
held by simple relatively low-energy hydrogen-bonding, whereas
in Na+-heparin water is held by stronger hydrogen bonds (Grant
et al., 1987a).
Because of the relationship suggested in Fig. 2, and because of
the apparent involvement of carboxylate groups in cation binding, the frequency at which the carboxylate band occurred for
various bivalent cation-heparin complexes was plotted against
the maximal change in optical rotation seen when high [cation]
was added to heparin (Fig. 5). The graph includes a plot of the
frequency measured in the presence of tetramethylammonium
ion, which binds minimally to heparin (Dais et al., 1988). For
four cations, an apparently linear relationship suggested by the
plot accords with the idea of the importance of the carboxylate
group in heparin-cation binding. The Mg2+-heparin complex
generates a point that diverges from the main part of the graph.
This result accords with the report that the 'two-variable' version
of the ion electrostatic condensation model (Manning, 1977,
1978) accounts adequately for Mg2+-heparin binding, but that
other multidentate cations, including Ca2+, bind to heparin more
strongly than predicted by the electrostatic model (Mattai &
Kwak, 1981, 1988). The behaviour of Mg2+ in this context
presumably results from the chemically 'hard' nature and
particular ionic radius of the Mg2+ ion, which restricts its
octahedral co-ordination to six poorly exchangeable water molecules of hydration.
In summary, our results suggest that the association between
Ca2' and heparin cannot be described adequately in terms of
simple electrostatic interactions. We believe that, in an aqueous
environment, some heparin-cation complexes may resemble
hydrated mineral-like colloidal states that are not subject to
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Received 18 March 1991/24 June 1991; accepted 24 July 1991
1992